Recent efforts in this laboratory have significantly improved the in vitro system for the functional reconstruction of rough microsomes in order to study the molecular events involved in the translocation of nascent pancreatic secretory proteins across the microsomal membrane. Using dog pancreas mRNA, micrococcal nuclease treated microsomal membranes, purified ribonuclease inhibitor and oxidized glutathione, we will now determine the function of proteolytic processing in the biogenesis of secretory proteins, the location of the transport peptide translocase among intracellular organelles, and the possible role of the large ribosomal subunit in maintaining the assembly of the membrane associated translocase during periods of active protein synthesis. We will examine the hypothesis that secretory proteins are exclusively translated on membrane bound ribosomes and use a lipid soluble alanine chloromethyl ketone derivative in an attempt to isolate the transport peptidase from dog pancreas microsomal membranes. Radioactive protein sequencing studies will be continued in order to complete the NH2 terminal sequences of seven pancreatic presecretory proteins (two forms of pretrypsinogen, two forms of prechymotrypsinogen, preproelastase, preprocarboxypeptidase A1 & preamylase). Two new areas will be explored: (1) The hypothesis of the enteropancreatic circulation of exocrine pancreatic proteins will be examined by tracing the fate of 35S met. labeled guinea pig pancreatic secretory proteins introduced into the blood circulation of the rat. Radioactivity appearing in pancreatic juice and bile will be analyzed by two dimensional IEF/SDS gel electrophoresis. (2) The character and time course of change in the synthesis of individual pancreatic secretory proteins, induced by nutritional factors and hormones, will be studied. Changes in protein synthesis with hormonal stimulation appear to be regulated at the level of translation. We will attempt to identify intracellular mediators cGMP, cAMP, calcium polyamines) which may be responsible for such changes.